Blood filtration methods

ABSTRACT

Methods wherein a blood cell concentration gradient is formed in a pooling unit in which blood is pooled before or after introducing the blood into a filter for eliminating leukocytes followed by the filtration are disclosed. By the first method comprising forming a blood cell concentration gradient in the pooling unit before introducing blood into the filter for eliminating leukocytes followed by the filtration, leukocytes can be efficiently eliminated from a whole blood preparation or a high platelet collection ratio can be established while maintaining a high leukocyte elimination ratio. By the second method comprising forming a blood cell concentration gradient in the pooling unit after introducing blood into the filter for eliminating leukocytes followed by the filtration, leukocytes can be efficiently eliminated from a whole blood preparation or a platelet preparation and platelets can be collected at a high ratio.

TECHNICAL FIELD

The present invention relates to a blood filtration method and a bloodfiltration apparatus for removing leukocytes from blood using aleukocyte-removing filter.

BACKGROUND ART

Because of recent progress in the blood transfusion medical science,blood component transfusion that transfuses only components necessaryfor a recipient is mainly employed in the present blood transfusionmedical treatment. Advantages such as a relieved burden on a recipientand an increased curing effect are given as the reasons for wideacceptance of the blood component transfusion. Blood component products,which are used for the transfusion, such as a concentrated erythrocyteproduct, concentrated platelet product, and blood plasma product, areprepared by centrifuging a whole blood product obtained by blooddonation. A large amount of leukocytes is contained in blood productsprepared in this manner. It has been discovered that such containedleukocytes induce comparatively slight side effects, such as headache,nausea, chills, and an non-hemolytic febrile transfusion reaction, aswell as serious side effects, such as alloantigen sensitization, virusinfection, and post-blood transfusion GVHD that seriously affect arecipient. For this reason, leukocyte-removing filters packed with afilter material such as a fiber material or porous material containingcontinuous pores has been widely used.

Removal of leukocytes using a leukocyte-removing filter includes thecase where the leukocytes are removed from a whole blood product and thecase where the leukocytes are removed from each blood component productthat has been prepared. In the latter case, a filter is required foreach blood component product. On the other hand, the former case isregarded to be more preferable, since leukocytes are removed from awhole blood product and the whole blood product from which theleukocytes have been removed is centrifuged to produce different andseveral kinds of blood component products not containing leukocytes.

Particularly, in recent years, a closed system prepared by integrating abag for whole blood products, filters, bags for various blood componentproducts that are obtained after centrifugal separation, and the like,to produce blood component products from which leukocytes have beenaseptically removed has attracted attention and is used at blood centersand the like (Japanese Patent Application Laid-open No. 320064/1989,etc.).

In the method for preparing blood free from leukocytes using a filtersuch as a closed system commonly employed in blood centers and the like,a system in which bags for blood containing a large amount ofleukocytes, a filter, and bags for collecting filtered blood, and thelike are connected with each other via soft tubes made from vinylchloride or the like may be used. More specifically, bags containingblood still containing un-removed leukocytes are hung on a rack withhooks and the like for a head drop of about 0.5–2 m, where the bloodcontaining un-removed leukocytes is thoroughly mixed to homogenize.Thereafter, a clamp or a breakable seal in the tube is manually releasedto collect blood from which leukocytes have been removed in the recoverybag through a filter. The operation is substantially continuouslyconducted to minimize a burden on filtration operators.

A currently commercially available closed system for filtering a wholeblood product removes leukocytes and plasma. Therefore, two bloodcomponent products from which leukocytes have been removed, aconcentrated erythrocyte product and a plasma product, can be eventuallyobtained.

Since a whole blood product contains a large amount of leukocytes, alarge capacity is required for the filter to remove leukocytes to thesame degree as that achieved when removing leukocytes from each bloodcomponent product. However, an increase in the filter capacity resultsin an increase in the amount of blood that remains in the filter. Sincethe remaining blood is discarded together with the filter afterfiltration, an increased filter capacity has a problem of an increase inthe loss of precious blood.

In addition, usually only a fresh whole blood product within three daysat most and in many cases within several hours after collection isfiltered for separation. Leukocytes removing capability of a filter isknown to decrease when filtering a whole blood product that is veryfresh and has a high temperature, in particular, within one hour aftercollection.

In view of these problems remaining still to be solved for efficientlyremoving leukocytes from a whole blood product, establishment of a bloodfiltering method that can remove leukocytes efficiently and at a highrate has been strongly desired.

In addition, a functional filter that can selectively remove onlyleukocytes from a whole blood product and recover platelets togetherwith erythrocyte and plasma is being developed in recent years(Transfusion vol. 39 (1999), No. 10S, Supplement, S541-040K, S542-040K).

Three blood component products from which leukocytes have been removed,a concentrated leukocyte-free erythrocyte product, concentrated plateletproduct, and plasma product, can be eventually obtained by filtrating awhole blood product using such a filter. However, since plateletsincluded in a fresh whole blood product are slightly activated due to astress or the like during blood collection and are caused to easily toadhere to a filter, the technology cannot attain the level to ensure ahigh platelet recovery rate in a stable manner at the present time.

Japanese Patent No. 2521090 discloses a filter in which the filtermaterial is maintained under wet conditions of saturated water contentor more with an aqueous solution of a water-soluble substance harmlessto water or living bodies. This technology reduces adhesion of plateletsby wetting the filter material with an aqueous solution such as aphysiological saline solution and increases the platelet recovery rate.However, according to investigations by the inventors of the presentinvention, a satisfactory increase in the platelet recovery rate was notachieved while maintaining high leukocyte removal capability. Inaddition, since the above disclosed filter is previously wetted with aphysiological saline solution or the like, air that may be accidentallyintroduced in the filter not only can be discharged to outside theapparatus only with difficulty, but also may decrease or suspend theblood flow.

A method of fractionating a whole blood product into blood componentproducts by centrifugation and filtering each blood component product isalso known. For example, Japanese Patent Application Laid-open No.2000-334034 discloses a method of subjecting a whole blood product to avery strong centrifugation of 3,520×g to separate the blood into threecomponents, plasma, a buffy coat with a large platelet content, andconcentrated erythrocytes, and sequentially filtering the buffy coat andconcentrated erythrocytes using one filter. Two component blood productsfrom which leukocytes have been removed, one a concentrated plateletproduct and the other a concentrated erythrocyte product, and a plasmaproduct from which leukocytes have not been removed can be ultimatelyprepared using this method. However, according to investigations of thepresent inventors, such strong centrifugation activates platelets makingit difficult to recover platelets at a satisfactory yield. In addition,the plasma product has a problem not to be removed leukocytes.

WO 92/07656 discloses a method and system of separating a whole bloodobtained by blood collection into a concentrated erythrocyte product andplatelet-rich plasma by weak centrifugation and filtering theseseparated fractions through different filters. Although leukocytes canbe removed from all ultimately obtained blood component productsaccording to the method, this method requires a complicated procedure ofinserting two filters together with a plurality of bags into acentrifuge cup. In addition, use of two filters increases the cost.

A filter for selectively removing leukocytes from a whole blood productor a platelet product, such as platelet-rich plasma product or aconcentrated platelet product prepared by a blood component collectionapparatus, is commercially available and used in blood centers orclinical work front. Although some portion of platelets having highadhesion can be recovered by using this filter, the platelet recoveryrate varies depending on individual difference of bloods and the like.Therefore, stability of the platelet recovery rate is still to beimproved.

In this manner, there are many subjects to be improved in the existingtechnology for removing leukocytes from a whole blood product or thetechnology for recovering platelets from a whole blood product or aplatelet product originating from a whole blood product or bloodcomponent collection while removing leukocytes. Development of a bloodfiltration method having an improved leukocytes removing capability sothat a need for increasing filter capacity may not be required,exhibiting an increased recovery rate of platelets if the platelets arerecovered, and being easily operated has been desired.

Since leukocytes removal has been recognized to be effective for safetransfusion, the amount of blood processed for removing leukocytes inblood centers and the like is increasing year by year. Automatic bloodfiltration has been desired to relieve burden loaded on operatorsprocessing a large amount of blood. Development to satisfy thisrequirement is ongoing.

Japanese Patent Application Laid-open No. 10-212237 discloses anapparatus for automatic filtration in which blood is filtered at aconstant flow rate by using a pump. The apparatus maintains anappropriate flow rate of the filtered blood while monitoring pressurenear the filter inlet port and controlling operation of the pumpaccording to the pressure.

Japanese Patent Application Laid-open No. 09-108334 discloses anapparatus that can detect the fact that a predetermined amount of bloodhas been processed using a timer, a sensor to optically detect the bloodsurface or the gas/blood interface, a weight sensor to detect the amountof blood, a pressure sensor to detect the pressure of a tube, and thelike, to automatically suspend filtration.

An automatic filtration apparatus in which not only is the bloodfiltration process automated, but also the blood filtration data(filtration time, temperature, flow rate, etc.) can be recoded by acomputer has been developed to satisfy the requirements for qualitycontrol of blood products that has become more stringent than ever (VoxSanguinis Vol. 78 (Supplement 1), 2000, P517, P518). An excellentfeature of the apparatus is its capability of simultaneouslyautomatically filtering 40 bloods while automatically homogenizing thebloods before or even during filtration by a mechanical means, and thecapability of automatically recording filtration data, and the like.

The above automatic apparatuses have been developed with an objective ofautomatically filtering blood in a homogeneous state, automaticallyfiltering at a constant flow rate of blood, and automatically suspendingfiltration operation. The blood filtration apparatus of the presentinvention is an automatic filtration initiate equipment suitable forimplementing a blood filtration method for improving performance of thefilter of the present invention and differs from the above-describedautomatic filtration apparatuses.

The features of the blood filtration method and automatic filtrationapparatus of present invention will be described in more detail.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic drawing showing an embodiment of the bloodfiltration apparatus of the present invention.

FIG. 2 is a schematic drawing showing another embodiment of the bloodfiltration apparatus of the present invention.

FIG. 3 is a block diagram of a control system of the blood filtrationapparatus shown in FIGS. 1 and 2.

FIG. 4 is a partial side view of an embodiment of a sensor and a storingunit, wherein an optical sensor is used as a signal transmitting means.

FIG. 5 is a partial side view of another embodiment of a sensor and astoring unit, wherein an optical sensor is used as a signal transmittingmeans.

FIG. 6 is a partial side view of still another embodiment of a sensorand a storing unit, wherein an optical sensor is used as a signaltransmitting means.

DISCLOSURE OF THE INVENTION

A first object of the present invention is to provide a blood filtrationmethod that can exhibit improved filtration performance and is easy tobe operated. More specifically, the present invention provides a bloodfiltration method that can efficiently remove leukocytes from a veryfresh whole blood product without increasing the filter capacity or canstably achieve a high platelet recovery rate while maintaining a highleukocyte removing capability when processing a whole blood product or aplatelet product derived from a whole blood product or a blood componentcollection. As a result of extensive studies, the inventors of thepresent invention have found that the above first object can be achievedby a method of filtering a blood that has been brought into aninhomogeneous state in which a blood cell concentration gradient hasbeen formed in a storing unit for storing the blood represented by ablood bag.

A second object of the present invention is to provide an automaticfiltration initiate apparatus suitable for use in the blood filtrationmethod of the present invention. The inventors of the present inventionhave found that the above second object can be achieved by a bloodfiltration apparatus provided with a signal transmitting means that canemit signals after an appropriate blood cell concentration gradient hasbeen formed and an operation means that can automatically release a flowcontrol means based on the signals from the signal transmitting means.

The objects of the present invention can be achieved by the followingblood filtration methods and blood filtration apparatuses:

(1) a blood filtration method for removing leukocytes from bloodcomprising forming a blood cell concentration gradient in a storing unitfor storing the blood, filtering the blood through a leukocyte-removingfilter, and collecting the filtered blood in a recovery unit,

(2) the blood filtration method described in (1) above, wherein theblood cell concentration gradient is previously formed in the storingunit before filling the filter with blood,

(3) the blood filtration method described in (1) or (2) above, whereinthe blood cell concentration gradient is formed by allowing the blood tostand for a predetermined period of time,

(4) the blood filtration method described in (3) above, wherein theblood cell concentration gradient is formed by allowing the blood tostand for not less than 5 minutes to less than 300 minutes,

(5) the blood filtration method described in (1) or (2) above, whereinthe blood cell concentration gradient is formed by weak centrifugation,

(6) the blood filtration method described in (5) above, wherein theblood cell concentration gradient is formed by weak centrifugation at acentrifugal force of not less than 100×g to not more than 1,200×g fornot less than 0.3 minutes to not more than 10 minutes,

(7) the blood filtration method described in (5) or (6) above, whereinthe blood cell concentration gradient is formed by weak centrifugation,and then the blood is allowed to stand for a predetermined period oftime,

(8) the blood filtration method described in (1) above, wherein theblood cell concentration gradient is formed in the storing unit afterfilling the filter with blood,

(9) the blood filtration method described in (1) or (8) above, whereinthe blood cell concentration gradient is formed by controlling the bloodflow rate for a predetermined period of time,

(10) the blood filtration method described in (9) above, wherein theblood cell concentration gradient is formed by controlling the bloodflow rate not less than 5 minutes to less than 300 minutes,

(11) the blood filtration method described in any one of (1) to (10)above, wherein the blood is a fresh whole blood product,

(12) the blood filtration method described in any one of (1) to (11)above, wherein the blood is introduced into the leukocyte-removingfilter from a lower part of the storing unit after forming anerythrocyte concentration gradient so that the hematocrit value of theblood in the lower part of the storing unit is larger than thehematocrit value of the whole blood product in the storing unit that hasbeen uniformly homogenized,

(13) the blood filtration method described in (12) above, wherein thehematocrit value of the blood in the lower part of the storing unit isnot less than 1.05 times to less than 2.50 times the hematocrit value ofthe whole blood product in the storing unit that has been uniformlyhomogenized,

(14) the blood filtration method described in (12) or (13) above,wherein the hematocrit value of the blood in the lower part of thestoring unit is not less than 35% to less than 75%,

(15) the blood filtration method described in any one of (11) to (14)above, wherein the blood cell separation degree of the blood in thestoring unit after forming the blood cell concentration gradient is notless than 3% to less than 60%,

(16) the blood filtration method described in (8) above, wherein theblood is a platelet product,

(17) the blood filtration method described in any one of (1) to (16)above, wherein leukocytes are selectively removed and platelets areallowed to pass through,

(18) the blood filtration method described in any one of (1) to (17)above, wherein the blood is filtered through one leukocyte-removingfilter,

(19) in a blood filtration apparatus comprising at least a storing unitfor storing blood, a holding unit for holding the storing unit, aleukocyte-removing filter, a recovery unit for collecting blood filteredthrough the leukocyte-removing filter, a first connecting tubeconnecting the storing unit with the leukocyte-removing filter, and asecond connecting tube connecting the leukocyte-removing filter with therecovery unit,

the blood filtration apparatus characterized by further comprising aflow control means for controlling a blood flow installed in the firstconnecting tube and/or the second connecting tube, a signal transmittingmeans worked in conjunction with the flow control means, and anoperation means that can automatically release the flow control meansbased on the signals from the signal transmitting means,

(20) the blood filtration apparatus described in (19) above, whereinsignals are emitted from the signal transmitting means after the storingunit has been held by the holding unit and allowed to stand for apredetermined period of time,

(21) the blood filtration apparatus described in (20) above, whereinsignals are emitted from the signal transmitting means after the storingunit has been held by the holding unit and allowed to stand for not lessthan 5 minutes to less than 300 minutes,

(22) the blood filtration apparatus described in (19) above, whereinsignals are emitted from the signal transmitting means after the storingunit containing blood after weak centrifugation has been held by theholding unit and allowed to stand for a predetermined period of time,

(23) the blood filtration apparatus described in (22) above, whereinsignals are emitted from the signal transmitting means after the storingunit containing blood after weak centrifugation at a centrifugal forceof not less than 100×g to not more than 1,200×g for not less than 0.3minutes to not more than 10 minutes has been held by the holding unitand allowed to stand for a predetermined period of time,

(24) the blood filtration apparatus described in (19) above, whereinsignals are emitted from the signal transmitting means after the bloodhas been filled in the filter and the flow rate has been allowed to becontrolled for a predetermined period of time,

(25) the blood filtration apparatus described in (24) above, whereinsignals are emitted from the signal transmitting means after the bloodhas been filled in the filter and the flow rate has been allowed to becontrolled for not less than 5 minutes to less than 300 minutes,

(26) the blood filtration apparatus described in any one of (19) to (25)above, wherein signals are emitted from the signal transmitting meansafter the blood separation degree has reached not less than 3% to lessthan 60%,

(27) the blood filtration apparatus described in any one of (19) to (25)above, wherein the signal transmitting means is a timer, and

(28) The blood filtration apparatus described in any one of (19) to (26)above, wherein the signal transmitting means is an optical sensor.

The blood filtration method of the present invention will be describedin detail.

Blood cells contained in blood have a specific gravity varying accordingto the type of blood cells. Erythrocytes are known to have a specificgravity of about 1.08; granulocytes and monocytes, about 1.07;lymphocytes, about 1.06; and platelets and plasma, about 1.03. If bloodcontaining blood cells with different specific gravities is allowed tostand, a blood cell concentration gradient is formed according to thedifference of the specific gravities. This property of blood is utilizedin the blood filtration method of the present invention.

The first blood filtration method of the present invention comprisespreviously forming a blood cell concentration gradient in the storingunit to make the blood inhomogeneous, introducing the blood into aleukocyte-removing filter from the lower part of the storing unit, andthen filtrating (this blood filtration method is hereinafter referred toas “first filtration method”). More specifically, the first filtrationmethod of the present invention is suitable for filtering a whole bloodproduct and comprises, in an inside of a storing unit represented by ablood bag for storing the whole blood product, causing first bloodfraction containing a large amount of erythrocytes and leukocytes(granulocytes, monocytes) having a high specific gravity to precipitatein a lower part of a storing unit, causing a layer of second bloodfraction containing a large amount of leukocytes (lymphocyte) having alow specific gravity so as to come above the first blood fraction, andcausing third blood fraction containing a large amount of platelets andplasma to come above the second blood fraction, thereby previouslymaking the whole blood product in the storing unit inhomogeneous withrespect to the blood cell concentration, and introducing the blood to beprocessed into a leukocyte-removing filter from the lower part of thestoring unit in the order of blood fractions in which the content ofblood cells with a high specific gravity decreases. Further, the firstfiltration method of the present invention filters substantially allblood including in the storing unit using a single filter.

Normally, the blood is mixed to make the blood cell concentrationhomogeneous before filtering through the leukocyte-removing filter. Thereason for homogenizing the blood is to prevent any part in theeffective filtering area from being clogged up with micro aggregatescontained in the blood at the initial stage of filtration. Since themicro aggregates are larger and heavier than blood cells, theyprecipitate in the lower part of the storing unit in an inhomogeneousstate. If the blood in such an inhomogeneous state is filtered,precipitated micro aggregates come in contact with the filter at acomparatively early stage and, cause a part of the effective filteringarea to become clogged up at an early stage of the filtration. This mayunduly retard the filtration due to an uneven flow of blood andremarkably decrease the filter performance. Since the phenomenon hasbeen thought to easily occur in blood preserved for several days ormore, a method to start filtration after the blood has been homogenizedas much as possible has been preferred. If a concentrated erythrocyteproduct with a small plasma content in an inhomogeneous state isfiltered, erythrocytes with a high specific gravity precipitate to forma very viscous portion with a high hematocrit value (a volume percentageof erythrocytes in blood). When the blood is processed using a filter,this portion may not flow through the filter. If pressure is applied toforcibly filter the blood, the erythrocytes may be destructed byhemolysis. Because of these phenomena, filtration after making the bloodas homogeneous as possible has been thought to be desirable to ensure astable blood flow and constant leukocyte removing capability.

However, as a result of extensive studies using comparatively freshwhole blood products preserved at room temperature, the presentinventors have found that the above problems are not observed even if awhole blood product is filtered in the order of blood cell concentrationand a high content of erythrocytes and granulocytes is firstly filteredafter a blood cell concentration gradient is formed to make the state ofthe blood inhomogeneous, and, surprisingly, the leukocyte removingcapability is remarkably increased.

An additional surprising finding was a remarkable increase in a plateletrecovery rate, when such an in homogenized whole blood product wasfiltered through a filter possessing a platelet recovering function.

As the reason why a liquid flow ability was unexpectedly obtained in thepresent invention even if a blood cell concentration gradient has beenformed, a fresh whole blood product that has been preserved for only avery short period of time after collection has a small content of microaggregates, and is thought not to cause the above-described problems dueto micro aggregates. In addition, a large plasma content of a wholeblood product as compared with a concentrated erythrocyte product makesit difficult to form unduly high viscous portions. This is supposed thata sufficient liquid flow ability has been acquired. However, thesignificant improvement in the leukocyte removal capability and theremarkable increase in the platelet recovery rate were unexpectedeffects. Although the mechanism for the change in the filtrationperformance due to in homogenization of a whole blood product is notknown, the followings are thought to be possible reasons. When a wholeblood product is in homogenized to cause erythrocytes and leukocyteswith a higher specific gravity to be precipitated in the lower part ofthe storing unit, the blood fraction in this lower part contains asmaller amount of plasma. Since the filter material at the initial stageof filtration is not sufficiently covered with plasma proteins thatsuppress adhesion of blood cells such as albumin, when such blood isprocessed through a filter, the number of leukocytes caused to adhere tothe surface of the filter material will increase, giving rise toimproved leukocyte removing capability. On the other hand, plateletspossessing a lower specific gravity are abundant in the upper layer ofthe inhomogenized whole blood product. When the platelets in the upperlayer are caused to come in contact with the filter material, thesurface of the filter would have already been covered with plasmaproteins such as albumin, making it difficult for the platelets to becaused to adhere to the surface. This results in an increased plateletrecovery rate.

The present inventors have further found that the platelet recovery ratecan be greatly increased by the second filtration method comprisingfilling blood in a filter having a platelet recovering function,controlling the blood flow in such a manner that the flow rate ismaintained at a low level or the blood flow is suspended for apredetermined period of time, and then filtering the blood (hereinafterreferred to as “second filtration method”).

More specifically, “the second filtration method” is suitable forprocessing a whole blood product or a platelet product such as aconcentrated platelet product and platelet-rich plasma to removeleukocytes while allowing platelets to pass through and comprisesfilling a filter with homogeneous blood, controlling the blood flow bymaintaining a low flow rate or suspending the flow for a predeterminedperiod of time, causing a blood cell concentration gradient to be formedin the blood in a storing unit while said controlling the blood flow,releasing the flow rate control means to start filtration, andcollecting the filtered blood from which leukocytes have been removed ina recovery unit such as a recovery bag. Both “the first filtrationmethod” and “the second filtration method” employ one filter to processsubstantially all blood containing in a storing unit.

There are following two possible reasons for the remarkable increase inthe platelet recovery rate by “the second filtration method”. One is thesame effect due to the blood cell concentration gradient formation as inthe first filtration method. The flow control operation for a specificperiod of time after filling the filter with blood caused a blood cellconcentration gradient to be formed in the blood in the storing unit.The other is the effect of plasma proteins such as albumin covering allover the surface of the filter material wetted with the blood during theflow control operation. The plasma proteins covering the surface of thefilter material may make it difficult for platelets to be caused toadhere to the surface.

The term “fill the filter with blood” as used in the second filtrationmethod in the present invention refers to an operation starting from thetime when blood is introduced into the filter from the filter inlet portuntil the time when the blood comes out from the outlet port. Since itis desirable for increasing the platelet recovery rate to wet the entirefilter with blood in said operation, the blood should be filled in thefilter so that air does not remain in the filter as much as possible.

The term “controlling a flow rate” in the present invention indicatesthe operation of maintaining the blood flow rate at a low level or ofsuspending the flow of blood. Here, “maintaining the blood flow rate ata low level” indicates the conditions in which almost no blood flows,specifically, at a flow rate in terms of average linear velocity in therange of more than 0 cm/min to not more than 0.2 cm/min, and preferablymore than 0 cm/min to not more than 0.1 cm/min, and “suspending theblood flow rate” indicates the conditions in which no blood flows,specifically, a flow rate in terms of average linear velocity of 0cm/min. The term “average linear velocity” described above refers to avalue obtained by dividing a blood flow rate (ml/min) by an effectivefiltering cross-sectional area (cm²) of filter perpendicular to thedirection of blood flow through which the blood can flow. A filter mayinclude a plurality of filter materials with different effectivefiltering cross-sectional areas depending on the configuration of thefilter. In such a case, an average of these effective filteringcross-sectional areas of the filter materials is used. In the presentinvention, either the control method of maintaining a low flow rate orthe control method of suspending the blood flow can be employed afterthe filter has been filled with blood. Suspending the blood flow is morepreferable due to simplicity of operation and the higher plateletrecovery rate.

The flow rate control of the present invention can be carried out usinga means functioning to retard or suspend the blood flow, for example,clamps such as a roller clamp, slide clamp, or Robert clamp, jigs thatcan deform a connecting tube by compression such as a forceps, and pumpsthat can control the linear velocity of blood. When using a jig that cancompressively deform a connecting tube, the flow rate can be controlledat a low level by decreasing the diameter of the opening port bycompressing the connecting tube or can be suspended by completelyblocking the connecting tube.

“The first filtration method” and “the second filtration method” of thepresent invention are particularly suitable when the blood to beprocessed is a whole blood product. A whole blood product herein refersto a blood product processed by an artificial means such as an additionof an appropriate amount of an anticoagulant to collected whole blood.There are no specific limitations to the amount of the whole bloodproduct to be filtered. More specifically, a whole blood product is ablood within three days, preferably within one day, and more preferablywithin eight hours after collection that contains an anticoagulant suchas ACD (acid citrate dextrose) or CPD (citrate·phosphate·dextrose). Inaddition, the whole blood product is preferably preserved at atemperature of not less than 4° C. to less than 30° C., preferably notless than 15° C. to less than 25° C., until the product is filteredafter collection. A whole blood product preserved for over three daysand/or a whole blood product preserved at a temperature of 4° C. or lesscontains an increased amount of micro aggregates. Such a whole bloodproduct may clog the filter with micro aggregates when inhomogenized,unpreferably resulting in an inadequate flow of blood. If preserved at atemperature above 30° C., unpreferably plasma proteins may easily bedenatured.

The whole blood product having a blood cell concentration gradient usedin “the first filtration method” and “the second filtration method” willbe described in detail. It is preferable that the whole blood producthaving a blood cell concentration gradient have a hematocrit value inthe lower part of the storing unit not less than 1.05 times to less than2.50 times as much as the hematocrit value of the whole blood product inthe storing unit that has been homogenized. If the hematocrit value inthe lower part of the storing unit is less than 1.05 times as much asthe hematocrit value of the blood that has been homogenized, formationof the blood cell concentration gradient is insufficient, whereby theleukocyte removing capability and platelet recovery rate tend not to beimproved. If more than 2.50 times, an increase in the filtration time,hemolysis due to an increased pressure loss, and a remarkable decreasein the flow rate may occur because of the increased blood viscosity. Ahematocrit value in the lower part of the storing unit is preferably notless than 1.05 times to less than 2.00 times, and particularlypreferably not less than 1.20 times to less than 1.70 times, as much asthe hematocrit value of the whole blood product that has beenhomogenized.

The hematocrit value of the whole blood product having a blood cellconcentration gradient in the lower part of the storing unit ispreferably not less than 35% to less than 75%. If the hematocrit valueis less than 35%, formation of the blood cell concentration gradient isinsufficient, whereby the leukocyte removing capability and plateletrecovery rate may not be improved. If more than 75%, an increase in thefiltration time, hemolysis due to an increased pressure loss, and aremarkable decrease in the flow rate may be induced. The more preferablerange is not less than 45% to less than 65%.

The hematocrit value in the lower part of the storing unit is preferablymeasured on 2–3 ml of a sample directly collected from the lower part ofthe storing unit. When direct sample collection is difficult, first 2–3ml of blood introduced into the tube connected to the storing unit whenthe whole blood product is filtered may be sampled and measured. Thehematocrit value can be measured by a conventional method such as acentrifugal method (e.g. microhematcrit method), electric conductivitymeasurement, pulse wave height method, or measurement using an automaticblood cell counter.

The whole blood product having a blood cell concentration gradient usedin “the first and second filtration methods” of the present inventionpreferably has a blood cell separation degree of not less than 3% toless than 60%, and more preferably not less than 10% to less than 45%.The blood cell separation degree used herein is a percentage of a liquidphase substance substantially not containing erythrocytes and containingplasma, platelets, and lymphocytes with a low specific gravity as maincomponents in the total amount of the blood. If the blood cellseparation degree is less than 3%, formation of the blood cellconcentration gradient is insufficient, whereby the filter performanceis not improved. If more than 60%, the hematocrit value in the lowerpart of the storing unit is excessively high, and an increase in thefiltration time, hemolysis due to an increased pressure loss, and aremarkable decrease in the flow rate may be induced.

As the method for forming a blood cell concentration gradient in thewhole blood product in “the first filtration method” in the presentinvention, a stationary method of allowing the blood preserved in thestoring unit to stand and a centrifugal method of centrifuging thestoring unit containing the whole blood product can be given.

To form a blood cell concentration gradient by the stationary method,the blood is preferably allowed to stand for not less than 5 minutes toless than 300 minutes. If less than 5 minutes, a sufficient blood cellconcentration gradient may not be formed; if 300 minutes or more, theperiod of time allowed to stand may be excessively long, and the bloodbecomes difficult to be filtered. The more preferable period of time isnot less than 10 minutes to less than 180 minutes, with a still morepreferable time being not less than 30 minutes to less than 120 minutes.

To form a blood cell concentration gradient by centrifugation, weakcentrifugation is preferable. For forming a blood cell concentrationgradient by weak centrifugation, the blood is preferably centrifuged,except for the period of acceleration and deceleration, at a centrifugalforce of about not less than 100×g to not more than 1,200×g, andespecially preferably not less than 100×g to not more than 500×g, for0.3 to 10 minutes. If the centrifugal force is less than this range,formation of the blood cell concentration gradient is insufficient. Ifmore than this range, the high hematocrit value in the lower part of thestoring unit may become high, and induce an increase in the filtrationtime due to increased viscosity. In addition, when the blood is filteredat a constant flow rate, a pressure loss may increase, giving rise tohemolysis of erythrocytes.

The whole blood product having a blood cell concentration gradient usedin “the first filtration method” of the present invention can beprepared by either the above-described stationary method or thecentrifugal method. The stationary method is more preferable due to thesimple operation. When it is desirable to allow platelets to passthrough, the centrifugal method tends to exhibit a somewhat small effecton the improvement in platelet recovery rate as compared with thestationary method, if the blood is filtered immediately aftercentrifugation. This is because platelets are somewhat activated by astress of centrifugation. For this reason, a stationary period ispreferably provided for a predetermined period of time after forming theblood cell concentration gradient by the centrifugal method to cause theactivated platelets to become deactivated. The stationary period fordeactivating the platelets is preferably not less than 5 minutes to lessthan 180 minutes, and more preferably not less than 10 minutes to lessthan 90 minutes.

In “the second filtration method” of the present invention, afterfilling the filter with the blood, the flow rate is controlled for apredetermined period of time, during which a blood cell concentrationgradient is formed in the storing unit. The flow rate control period ispreferably not less than 5 minutes to less than 300 minutes. If the flowrate control period is less than 5 minutes, the blood cell concentrationgradient in the storing unit may be insufficiently formed. In addition,improvement of the platelet recovery rate may not be observed due todifficulty of a sufficient coverage of the filter material with plasmaproteins. A flow rate control period of 300 minutes or more isunpreferably too long for the time controlling the flow rate to filter alarge quantity of blood. The more preferable period of time is not lessthan 10 minutes to less than 180 minutes, with a most preferable timebeing not less than 30 minutes to less than 120 minutes.

“The second filtration method” of the present invention is effective notonly when the blood to be filtered is a whole blood product, but alsowhen a platelet product such as a concentrated platelet product andplatelet-rich plasma product is filtered.

The platelet product such as a concentrated platelet product orplatelet-rich plasma product used herein refers to a platelet productprepared by centrifuging collected whole blood product or a plateletproduct originating from a blood component collection. A plateletproduct within five days after preparation is preferable. In addition,such a platelet product is preferably preserved while shaking at roomtemperature of not less than 20° C. to less than 25° C. until theproduct is filtered after preparation. A platelet product preserved formore than five days or at a temperature outside of the above temperaturerange is not preferable due to the possibility of impaired plateletfunction.

A platelet product having a blood cell concentration gradient refers toa product having a leukocyte concentration in the lower part of thestoring unit 1.05 times or more of the leukocyte concentration of theblood which has been homogenized. If less than 1.05 times, leukocyteremoving capability tends to decrease. A more preferable range is notless than 1.20 times to less than 2.50 times.

The leukocyte concentration in the lower part of the storing unit can bemeasured by dying leukocyte nuclei and observing the dyed leukocytenuclei using a microscope. In this instance, 2–3 ml of a blood sample iscollected directly from the lower part of the storing unit or from 2–3ml of a blood sample initially introduced into the tube connected to thestoring unit.

The blood filtration method of the present invention is preferablycarried out using the storing unit for storing blood, the recovery unitfor collecting filtered blood, and hollow tubes connecting the storingunit/recovery unit with the filter. A sterile connecting device (SCD)may be used for connecting with tubes. There are no specific limitationsto the tube used here inasmuch as the tube does not damage blood cells.Organic materials such as vinyl chloride, silicone, polysulfone,polyamide, polyester, urethane, polyethylene, and polypropylene arepreferable due to excellent formability.

Any container that can store blood and is made of a material notactivating blood cells or not inducing adhesion or denaturation ofplasma proteins can be used as the storing unit for storing bloodwithout specific limitations. Blood bags made of soft polyvinyl chlorideor polyolefin commonly used for collecting and storing blood andsyringes made of polypropylene, polyethylene, or polystyrene can begiven as more specific examples.

Any container that can collect and store blood and is made of a materialnot activating blood cells and not inducing adhesion or denaturation ofplasma proteins can be used as the recovery unit without specificlimitations. The recovery unit may include at least one container forcollecting each component separated by centrifugation of filtered blood.Blood bags made of soft polyvinyl chloride or polyolefin commonly usedfor collecting and storing blood can be given as more specific examples.

As the leukocyte-removing filter of the present invention, any knownleukocyte-removing filter having inlet and outlet ports for blood andpacked with a filter material that can capture and remove leukocytes inblood can be preferably used. More specifically, such a filter is afilter possessing a leukocyte removing capability of 2.30 or more,preferably 3.00 or more, wherein the leukocyte removing capability isdefined as a logarithmic value of a value obtained by dividing theconcentration of leukocytes remaining after filtration by the leukocyteconcentration before filtration, i.e. −Log (leukocyte concentrationafter filtration/leukocyte concentration before filtration).

A fibrous medium, a sponge-like medium, and the like can be given as thefilter material. With an objective of making the filter material easilywetted with blood, the surface of the filter material may be modified bycoating a hydrophilic polymer or by radiation graft polymerization tothe extent that blood is not adversely affected.

A material with low adherence to platelets may be introduced to thesurface of the filter material to provide the leukocyte-removing filterwith a platelet permeating function. As the material with low adherenceto platelets, a polymer having a hydrophilic group and a chargeablegroup such as an amino group or carboxyl group and polyurethane asdescribed in Japanese Patent Publication No. 06-51060 and JapanesePatent Application Laid-open No. 01-249063 can be given as suitablematerials.

The blood filtration method of the present invention is a method forfiltering blood using a leukocyte-removing filter. The blood may befiltered after providing an appropriate head drop using a blood bag orthe like as a storing unit or may be filtered at a constant flow rateusing a pump or the like.

A second object of the present invention is to provide an apparatus forautomatically performing the blood filtration method such as “the firstfiltration method” or “the second filtration method” that requires anoperation of allowing the blood to stand or of controlling the flow ratefor a predetermined period of time before initiating blood filtrationusing a filter actually and to provide a blood filtration apparatus forperforming “the first filtration method” and “the second filtrationmethod” in stable filter performance while relieving burden loaded on anoperator. The blood filtration apparatus of the present invention willbe described in detail referring to the appended drawings. However, theblood filtration apparatus of the present invention is not limited tothe specific embodiments described in the drawings.

As shown in FIGS. 1 and 2, the blood filtration apparatus (1) of thepresent invention comprises a storing unit (2) for blood typified by ablood bag, a holding unit (11) for holding the storing unit (2) typifiedby a hook, a recovery unit (3), and a filter (4) having an inlet portand an outlet port for blood connected by hollow connecting tubes (5,6),and a flow rate controlling means (7,7′) in the connecting tubes, ofwhich the operation is automatically released by an operation means (10)based on signals emitted by signal transmitting means (8,9).

In the blood filtration apparatus (1) of the present invention, beforethe blood is actually processed through the filter (4), the blood flowis controlled at a low level or suspended by the flow rate controllingmeans (7,7′) in the first connecting tube (5) and/or the secondconnecting tube (6) for a predetermined period of time so that the bloodcannot substantially pass through. The blood flow control isautomatically released when the operating means (10) recognizes thelater-described signals emitted from the signal transmitting means (8,9)and sends the signals to the flow control means (7,7′), whereupon thefiltration starts. Specifically, the flow control means (7,7′), thesignal transmitting means (8,9), and the operating means (10) must workin conjunction with in the blood filtration apparatus (1) of the presentinvention. The term “work in conjunction with” herein refers to a statein which the signals from the signal transmitting means (8,9) can beautomatically sent to the flow control means (7,7′) via the operatingmeans (10). This means that the flow control means (7,7′), the signaltransmitting means (8,9), and the operating means (10) are electricallyconnected. The operating means (10) may be either a unit integrallyformed with the signal transmitting means (8,9) and/or the flow controlmeans (7,7′) or an independent unit.

The signal transmitting means is an instrument capable of emittingsignals to the operating means (10) when a predetermined period of timehas elapsed or when a predetermined blood cell concentration gradienthas been formed in the blood. As an example of the signal transmittingmeans (8) in FIG. 1, a timer that can release operation of the flowcontrol means (7,7′) after a predetermined period of time can be given.As a preferable example of the signal transmitting means (9) in FIG. 2,an optical sensor that can detect the state of formation of the bloodcell concentration gradient (i.e., the blood cell separation degree) inthe storing unit (2) and can work in conjunction with the flow controlmeans (7,7′) can be given.

The timer can be used either in “the first filtration method” or in “thesecond filtration method”. In FIG. 1, when a timer is used as the signaltransmitting means (8), the timer is set so that signals are emittedfrom the timer upon elapse of a predetermined period of time aftercausing the storing unit (2) to hold by the holding unit (11) and standstill or after the filter (4) has been filled with blood and the bloodflow rate has been controlled. The signals are sent to the flow controlmeans (7,7′) via the operating means (10) , whereupon the flow controlmeans (7,7′) are automatically released and filtration is initiated.

The optical sensor is a signal transmitting means that is particularlysuitable for use when a whole blood product is filtered. In a bloodfiltration apparatus as shown in FIG. 2, when an optical sensor is usedas the signal transmitting means (9) to filter a whole blood product,the optical sensor is set so that signals are emitted from the opticalsensor when the sensor detects that a predetermined blood cellseparation degree has been reached after the storing unit (2) has beencaused to hold by the holding unit (11) and stand still and the bloodcell concentration gradient has been formed. The signals are sent to theflow control means (7,7′) via the operating means (10), whereupon theflow control means (7,7′) are released.

As the optical sensor, a sensor optically detecting the location of ablood interface (for example, the interface of a layer containing alarge amount of plasma, platelets, and lymphocytes with a low specificgravity and a layer containing a large amount of erythrocytes,granulocytes and monocytes with a high specific gravity) when the bloodcell concentration gradient has been formed, as shown in FIG. 4, can begiven. The sensor contains a photo emitting element and a photoacceptance element. Light emitted from the photo emitting element isreflected by blood and the reflected light is received by the sensor.When blood separation proceeds and the blood interface reaches thelocation at which the sensor is installed, the intensity of reflectedlight changes. Utilizing this principle, the sensor is installed at thelocation corresponding to an appropriate degree of blood cell separationso that signals are emitted from the sensor at the time when theintensity of reflected light has changed, whereby the flow control means(7,7′) are automatically released to start blood filtration.

As another example of the optical sensor, a device comprising a sensorhaving a photo emitting element and a sensor having a photo acceptanceelement, installed on both sides of the storing unit (2), as shown inFIG. 5, can be given. This type of sensor detects the intensity oftransmitted light. When the blood interface reaches the location of thesensor, specifically when the intensity of transmitted light hasincreased, signals are emitted from the sensor, whereby the flow controlmeans (7,7′) are automatically released.

Alternatively, as shown in FIG. 6, sensors installed on the upper partand lower part of the storing unit can also be used in the presentinvention. The sensors are designed to emit signals when the differencein the intensity of reflected lights or transmitted lights from theupper part and lower part reaches a predetermined value, whereupon theflow control means (7,7′) are automatically released.

Any device provided with a function of maintaining the blood flow rateat a low level or of suspending the blood flow, cooperating with thesignal transmitting means (8,9) such as the above-described timer oroptical sensor, and capable of automatically releasing the blood flowcontrol based on the signals from the signal transmitting means (8,9)can be used without specific limitations as the flow rate controllingmeans (7,7′) in the blood filtration apparatus of the present invention.Examples include a clamp, valve, and pump. The term “releasing the flowrate controlling means (7,7′)” herein indicates, when the flow rate iscontrolled by compressing the connecting tube using a clamp or a valve,for example, expanding the opening diameter of the connecting tube toallow the blood to flow through. When the flow rate is controlled by apump, the term “releasing the flow rate controlling means (7,7′)”indicates initiating blood flow by operating the pump or increasing theblood flow rate by increasing the pump capacity.

Although the embodiments shown in FIGS. 1 and 2 are of the type in whichthe flow rate controlling means (7,7′) are installed both in the firstconnecting tube (5) connecting the storing unit (2) with the filter (4)and in the second connecting tube (6) connecting the filter (4) with theblood recovery unit (3), the flow rate controlling means can be providedto either one of the connecting tubes.

A blood bag made of a soft material, if used as the storing unit (2),may be deformed and result in unstable detection of the interface by theoptical sensor. A holder to stabilize the blood bag in a stableconfiguration can be provided to overcome this problem and to improvethe precision of the sensor.

Any type of holder may be used as the holding unit (11) to hold thestoring unit (2) of the present invention without a specific limitationto the extent that the holder can function to hold the storing unit in apredetermined position. A holder that can hold the storing unit standingstill without causing vibration is particularly preferable. A hook orthe like on which the storing unit is hung to provide a desired headdrop can be given as a more specific example.

There are no specific limitations to the manner of filtration using theblood filtration apparatus (1) of the present invention. Afterautomatically releasing operation of the flow rate controlling means(7,7′) by the signal transmitting means (8,9) such as a timer or anoptical sensor, blood may be filtered either by an appropriate head dropin the range of about 0.5–2 m or by a pump cooperating with the signaltransmitting means (8,9).

When a whole blood product is filtered using the blood filtration methodand/or blood filtration apparatus of the present invention, it ispossible to prepare leukocyte-free blood component products from thewhole blood product, from which leukocytes have been removed, by a knowncentrifugal method.

BEST MODE FOR CARRYING OUT THE INVENTION

The blood filtration method and blood filtration apparatus of thepresent invention will now be described in detail by way of examples,which should not be construed as limiting the present invention.

Examples of the first blood filtration method are given at thebeginning.

EXAMPLE 1

(Leukocyte-Removing Filter)

The experiment was carried out using a nonwoven polyethyleneterephthalate fabric with an average fiber diameter of 1.2 μm, theweight of the substrate per unit area (Metsuke) of 40 g/m², and athickness of 0.2 mm, and a random copolymer of 2-hydroxyethylmethacrylate (HEMA) and dimethylaminoethyl methacrylate (DM) (mol ratioof HEMA:DM=97:3, hereinafter referred to as HM-3) as the material withlow adherence to platelets.

(Synthesis of Polymer and Coating)

HM-3 polymer was prepared by random copolymerization of the monomers inethanol (monomer concentration: 1 mol/l) in the presence of 1/200 mol/lof 2,2′-azobis(2,4-dimethylvaleronitrile) (V-65, manufactured by WakoPure Chemical Industries, Ltd.) as an initiator for eight hours at 60°C.

The HM-3 polymer thus synthesized was coated on the surface of thenonwoven fabric according to the following procedure. The synthesizedHM-polymer was dissolved in a mixed solvent of ethanol and water (weightratio, ethanol:water=70:30) to prepare a liquid with a concentration of7 g/dl. A specimen with a diameter of 25 mm punched from the nonwovenfabric was dipped in the polymer solution for one minute at 25° C. andfilled in a holder made of polycarbonate. Dry nitrogen was passed thoughthe holder for 4.5 minutes and the holder was dried under vacuum for 18hours at 60° C., thereby coating the nonwoven fabric with the polymer.

(Preparation of Filter)

A filter for filtering blood was prepared by punching the nonwovenfabric to obtain packing disks with a diameter of 20 mm and packing 16sheets of the packing disks in a container with an effective filtrationcross-sectional area of 1.33 cm² having a blood inlet port and outletport. When using the nonwoven fabric coated with HM-3, dry HM-3 coatednonwoven fabric was taken off from the polycarbonate holder and punchedto obtain packing disks with a diameter of 20 mm. 16 packing disks thusobtained were stacked and packed.

(Blood Filtration Test: First Filtration Method)

A CPD-added whole blood product collected in a soft polyvinyl chlorideblood bag and preserved at room temperature (20–25° C.) for 2–3 hourswas sufficiently homogenized. 8 ml of the blood sample was collectedusing a syringe (brand name: Terumo Syringe® SS-20ESZ, manufactured byTerumo Corp.) from the homogenized whole blood product. The syringecontaining the whole blood product was connected with a polyvinylchloride tube with an internal diameter of 2.5 mm equipped with a clamp.The other end of the tube was connected with the blood inlet port of theabove-described filter. The same tube was connected with the bloodoutlet port of the filter, with the other end of the tube being providedwith a spit tube made of polyethylene for collecting blood dischargedtherefrom. Thereafter, a clamp provided between the syringe containingthe whole blood product and the filter inlet port closed the, tube.After allowing the vertically placed syringe to stand for 60 minutes,the clamp was released and blood was filtered at a constant flow rate of0.9 ml/min.

(Determination of Leukocyte Removing Capability and Platelet RecoveryRate)

The leukocyte concentration in a whole blood product before filtrationwas measured by an optical microscope after dying leukocytes in theblood sample collected from a homogeneous whole blood product with aTürk fluid. The leukocyte concentration in the whole blood product afterfiltration was measured by a fluorescence microscope after sampling ablood collected in the polyethylene spit tube and dying leakingleukocytes with an acridine orange solution. The leukocyte removingcapability was determined from the leukocyte concentrations before andafter the filtration according to the following formula.Leukocyte removing capability=−Log(leukocyte concentration afterfiltration/leukocyte concentration before filtration)

For calculating the platelet recovery rate, the platelet concentrationsbefore and after the filtration were determined using the same bloodsample as the blood measured leukocyte concentration by a multi-purposeautomatic blood cell counter (K-4500, manufactured by Sysmex Corp.). Theplatelet recovery rate was calculated according to the followingformula.Platelet recovery rate=(platelet concentration after filtration/plateletconcentration before filtration)×100 (%)(Determination of Hematocrit Value)

The hematocrit value of homogeneous blood (hereinafter referred to asPre-Ht) and the hematocrit value at a lower part of the syringe(hereinafter referred to as Post-Ht) were determined as follows. Todetermine Pre-Ht, the whole blood product before processing with thefilter remaining in the blood bag was sufficiently homogenized and 2 mlof sample blood was collected therefrom. Another similar syringecontaining a whole blood product was prepared and allowed to stand for apredetermined period of time and 2 ml of blood was sampled from thelower part of the syringe for use to determine the Post-Ht of a wholeblood product having a blood cell concentration gradient. In theComparative Example described later, the whole blood product allowed tostand for a predetermined period of time was again homogenized and 2 mlof blood was sampled from the lower part of the syringe for use in thePost-Ht determination. A blood sample was put into a polyethylene spittube and Pre-Ht and Post-Ht was determined using the same multi-purposeautomatic blood cell counter as used for the determination of theplatelet concentration.

As a result, Pre-Ht was 34%, Post-Ht was 52% (Post-Ht/Pre-Ht=1.53), theleukocyte removing capability was 3.72, and the platelet recovery ratewas 84%.

EXAMPLE 2

The same whole blood, product was filtered using the same filter as usedin Example 1 in the same manner as in Example 1, except that thestanding-still period of time was five minutes. As a result, Post-Ht was37% (Post-Ht/Pre-Ht=1.08), the leukocyte removing capability was 3.80,and the platelet recovery rate was 75%.

EXAMPLE 3

The same whole blood product was filtered using the same filter as usedin Example 1 in the same manner as in Example 1, except that thestanding-still period of time was 10 minutes. As a result, Post-Ht was41% (Post-Ht/Pre-Ht=1.21), the leukocyte removing capability was 3.89,and the platelet recovery rate was 81%.

EXAMPLE 4

The same whole blood product was filtered using the same filter as usedin Example 1 in the same manner as in Example 1, except that thestanding-still period of time was 180 minutes. As a result, Post-Ht was57% (Post-Ht/Pre-Ht=1.68), the leukocyte removing capability was 3.70,and the platelet recovery rate was 93%.

COMPARATIVE EXAMPLE 1

The same whole blood product as used in Example 1 was collected in asyringe and allowed to stand for 60 minutes. The blood was againhomogenized to obtain a homogeneous whole blood product substantiallyhaving no blood cell concentration gradient. The whole blood product wasfiltered using the same filter as used in Example 1 in the same manneras in Example 1. As a result, Post-Ht was 35%, and equal to the Pre-Ht(Post-Ht/Pre-Ht=1.00). The leukocyte removing capability was 3.14 andthe platelet recovery rate was 65%.

EXAMPLE 5

The experiment was carried out using a nonwoven polypropyleneterephthalate fabric with an average fiber diameter of 1.4 μm, a Metsukeof 42 g/m², and a thickness of 0.22 mm and a random copolymer of HEMAand diethylaminoethyl methacrylate (DE) (mol ratio of HEMA:DE=95:5,hereinafter abbreviated to HE-5) as the material with low adherence toplatelets. The HE-5 polymer was synthesized, the nonwoven fabric wascoated, and blood was filtered in the same manner under the sameconditions as in Example 1. As a result, the leukocyte removingcapability was 3.65 and the platelet recovery rate was 85%.

EXAMPLE 6

The experiment was carried out using a random copolymer of hydroxypropylmethacrylate (HPMA) and DM (mol ratio of HPMA:DM=97:3, hereinafterreferred to as PM-3) as the material with low adherence to platelets.The same nonwoven fabric was used as in Example 1, and PM-3 polymer weresynthesized, the nonwoven fabric was coated, and blood was filtered inthe same manner under the same conditions as in Example 1. As a result,the leukocyte removing capability was 3.74 and the platelet recoveryrate was 88%.

EXAMPLE 7

The experiment was carried out using a random copolymer of HPMA, DM, andmethoxydiethylene glycol methacrylate (DEG) (mol ratio ofHPMA:DM:DEG=90:3:7, hereinafter referred to as PMD) as the material withlow adherence to platelets. The same nonwoven fabric was used as inExample 1, and PMD polymer was synthesized, the nonwoven fabric wascoated, and blood was filtered in the same manner under the sameconditions as in Example 1. As a result, the leukocyte removingcapability was 3.79 and the platelet recovery rate was 90%.

Comparing the results obtained in Examples 1–7 and Comparative Example1, it can be seen that when a leukocyte-removing filter coated with amaterial with low adherence to platelets was used, not only theleukocyte removing capability, but also the platelet recovery rate wasremarkably improved by forming a blood cell concentration gradient inthe whole blood product.

EXAMPLE 8

A filter packed with the same nonwoven fabric as used in Example 1 notcoated with a material with low adherence to platelets was prepared. ACPD-added whole blood product, three hours after collection, wascollected in a syringe and allowed to stand for 60 minutes. The bloodwas filtered in the same manner as in Example 1. As a result, Pre-Ht was39%, Post-Ht was 62% (Post-Ht/Pre-Ht=1.59), and the leukocyte removingcapability was 4.34.

COMPARATIVE EXAMPLE 2

The same whole blood product as used in Example 8 was collected in asyringe and allowed to stand for 60 minutes. The blood was againhomogenized to obtain a homogeneous whole blood product substantiallyhaving no blood cell concentration gradient. The whole blood product wasfiltered using the same filter as used in Example 8 in the same manneras in Example 8. As a result, Post-Ht was 39%, and equal to the Pre-Ht(Post-Ht/Pre-Ht=1.00). The leukocyte removing capability was 3.51.

Comparing the results obtained in Example 8 and Comparative Example 2,it can be seen that when a leukocyte-removing filter not coated with amaterial with low adherence to platelets was used, the leukocyteremoving capability was remarkably improved by forming a blood cellconcentration gradient.

EXAMPLE 9

A nonwoven fabric with an average fiber diameter of 12 μm, a Metsuke of30 g/m², and a thickness of 0.2 mm (hereinafter referred to asprefilter), a nonwoven fabric with an average fiber diameter of 1.7 μm,a Metsuke of 66 g/m², and a thickness of 0.4 mm (hereinafter referred toas main filter A), and the same nonwoven fabric as used in Example 1(hereinafter referred to as main filter B) were coated with HM-3polymer. The following coating method was used. The nonwoven fabricswere cut into squares (length of one side: 10 cm) and were dipped in asolution dissolving HM-3 polymer at 25° C. for one minute. Afterremoving excessive HM-3 solution on the nonwoven fabrics by compressingthe nonwoven fabrics wetted with the HM-3 solution to a thickness of onehalf the wet thickness, the nonwoven fabrics were dried at roomtemperature for 24 hours, thereby coating the nonwoven fabrics with HM-3polymer. The procedure was carried out for each sheet of coated nonwovenfabric until all sheets required for packing in the filter wereobtained. A 5 g/dl HM-3 solution was used for coating the prefilter,whereas a 7 g/dl HM-3 solution was used for coating the main filters Aand B.

The coated nonwoven fabrics thus obtained (two sheets of prefilter, twosheets of main filter A, and 28 sheets of main filter B) were packed ina polycarbonate container with an effective filtration cross-sectionalarea of 45 cm² in the order of the prefilter, main filter A, and mainfilter B from the inlet port side to the outlet port side. An objectivefilter was then prepared by ultrasonic welding.

A CPD-added whole blood product (450 ml) collected in a soft polyvinylchloride blood bag and preserved at room temperature (22–24° C.) for 3hours. The blood bag was connected to the inlet port of the above filtervia a tube with an internal diameter of 2.9 mm to which a clamp wasattached. The outlet port of the filter was connected with the bloodrecovery bag using the same tube. The blood bag containing the wholeblood product was hung on a hook to provide a head drop of 1.0 m. Afterallowing to stand for 60 minutes, the clamp was released to collectfiltered blood in the blood recovery bag. The hematocrit value wasdetermined using a homogeneous blood sample and a sample collected froma lower part of the blood bag after the blood had been allowed to standstill for 60 minutes in the same manner as above. As a result offiltration, Pre-Ht was found to be 38%, Post-Ht was 54%(Post-Ht/Pre-Ht=1.42), the leukocyte removing capability was 4.12, andthe platelet recovery rate was 87%.

EXAMPLE 10

The filtration was carried out according to the procedure of Example 9with respect to the nonwoven fabrics, coating, filter, and method andconditions for filtration, except for using the same polymer with lowadherence to platelets as used in Example 7. As a result, the leukocyteremoving capability was 4.30 and the platelet recovery rate was 92%.

EXAMPLE 11

A fresh CPD-added whole blood collected in a soft polyvinyl chlorideblood bag and preserved at room temperature for three hours was put intoa centrifugal separator (CR7B3; manufactured by Hitachi Koki Co., Ltd.)and lightly centrifuged at 300×g of centrifugal force for five minutesto prepare a whole blood product (28 ml) having a blood cellconcentration gradient. A filter was prepared by stacking and packing 16sheets of nonwoven fabrics coated with HM-3 polymer, the same as thoseused in Example 1, in a holder with an internal diameter of 25 mm. Thebag containing the whole blood product, in which a blood cellconcentration gradient was formed by centrifugation, was connected witha polyvinyl chloride tube with an internal diameter of 2.5 mm. The otherend of the tube was connected with the blood inlet port of the filter.The outlet port of the filter was connected with the blood bag forcollecting the filtered whole blood product through the same tube.Pre-Ht was measured using a blood sample collected from homogeneouswhole blood product before centrifugation and Post-Ht was measured usinga blood sample collected from a lower part of the blood bag containing awhole blood product that had been allowed to stand for five minutesafter centrifugation. As a result, Pre-Ht was 32% and Post-Ht was 63%(Post-Ht/Pre-Ht=1.97).

After connecting with the filter, the blood bag containing the wholeblood product having a blood cell concentration gradient was suspendedto provide a head drop of 40 cm for filtration. The blood was filtered.As a result, the leukocyte removing capability was 3.48 and the plateletrecovery rate was 76%.

EXAMPLE 12

A fresh CPD-added whole blood product preserved for four hours at roomtemperature after collection was centrifuged in the same manner as inExample 11 and allowed to stand still for 60 minutes. Except for theabove procedure, the filtration was carried out using the same nonwovenfabrics, coating, and filter as used in Example 11 in the same manner asin Example 11. As a result, Pre-Ht was 37%, Post-Ht was 66%(Post-Ht/Pre-Ht=1.78) the leukocyte removing capability was 3.69, andthe platelet recovery rate was 82%.

Examples of the second filtration method will now be described.

EXAMPLE 13

The experiment for the second filtration method was carried out usingthe same nonwoven fabric, polymer with low adherence to platelets, andfilter as used in Example 1. The leukocyte removing capability andplatelet recovery rate were determined in the same manner as in Example1.

(Blood Filtration Test: Second Filtration Method)

A CPD-added whole blood product collected in a soft polyvinyl chlorideblood bag and preserved at room temperature (20–25° C.) for 2–3 hourswas sufficiently homogenized. 8 ml of a blood sample was collected fromthis product using a syringe (brand name: Terumo Syringe® SS-20ESZ,manufactured by Terumo Corp.). The syringe containing the whole bloodproduct was connected with a polyvinyl chloride tube with an internaldiameter of 2.5 mm. The other end of the tube was connected with theblood inlet port of the above-described filter. The same tube butequipped with a clamp was connected with the blood outlet port of thefilter. A soft polyvinyl chloride bag was arranged as a blood recoverybag to collect blood discharged from the other end of the filter. Thewhole blood product in a syringe was sufficiently homogenized and filledin the filter at a constant linear velocity of 0.68 cm/min (a flow rateof 0.9 ml/min). The pump was stopped when the blood came from the outletport of the filter. The tube was closed using the clamp at a pointbetween the filter and the recovery bag to suspend blood flow for 60minutes. Then, the clamp was released to run the pump again and filterthe blood at a constant linear velocity of 0.68 cm/min. As a result, theleukocyte removing capability was 3.58 and the platelet recovery ratewas 92%.

EXAMPLE 14

A whole blood product was filtered using the same filter as used inExample 13 in the same manner as in Example 13, except that the bloodflow was suspended for five minutes. As a result, the leukocyte removingcapability was 3.60 and the platelet recovery rate was 83%.

EXAMPLE 15

A whole blood product was filtered using the same filter as used inExample 13 in the same manner as in Example 13, except that the bloodflow was suspended for one minute. As a result, the leukocyte removingcapability was 3.62 and the platelet recovery rate was 76%.

COMPARATIVE EXAMPLE 3

A whole blood product was filtered using the same filter as used inExample 13 in the same manner as in Example 13, except that the bloodflow was not suspended after the blood was filled in the filter. As aresult, the leukocyte removing capability was 3.64 and the plateletrecovery rate was 64%.

COMPARATIVE EXAMPLE 4

The same filtration experiment as in Example 13 was carried out usingthe same filter as in Example 13, except that a physiological salinesolution was filled in the filter instead of the blood. As a result, theleukocyte removing capability was 3.50 and the platelet recovery ratewas 69%.

EXAMPLE 16

The filtration was carried out in the same manner under the sameconditions as in Example 13 using the same nonwoven fabric, polymer, andfilter as in Example 5. As a result, the leukocyte removing capabilitywas 3.48 and the platelet recovery rate was 93%.

EXAMPLE 17

A filtration experiment was carried out under the following conditionsusing the same filter as used in Example 9.

A CPD-added whole blood product (450 ml) collected in a soft polyvinylchloride blood bag and preserved at room temperature (22–24° C.) forfour hours was sufficiently homogenized. The blood bag was connectedwith the blood inlet port of the filter using a tube equipped with aclamp. The blood outlet port of the filter was connected with the bloodrecovery bag using the same tube. The bag containing the whole bloodproduct was located at a height to provide a head drop of 1.0 m. Theclamp was closed at the time when the filter was filled with ahomogeneous whole blood product to suspend the flow of blood for 60minutes. Then, the clamp was released to filter the blood. As a result,the leukocyte removing capability was 3.64 and the platelet recoveryrate was 95%.

EXAMPLE 18

The same filter as in Example 13 was filled with the same whole bloodproduct as in Example 13 at a constant linear velocity of 0.68 cm/min.Thereafter, the setting of the pump was changed to maintain a linearvelocity of 0.08 cm/min for five minutes. After setting back the linearvelocity to 0.68 cm/min by changing the setting of the pump again, theblood was filtered. As a result, the leukocyte removing capability was3.61 and the platelet recovery rate was 78%.

EXAMPLE 19

A filter with an effective filtration cross-sectional area of 1.33 cm²was prepared by packing 14 sheets of the nonwoven fabrics coated withHM-3 prepared in the same manner as in Example 13 in a container havingan inlet port and an outlet port. 8 ml of a concentrated plateletproduct, prepared by centrifuging a CPD-added whole blood product andpreserved for three days at 22–24° C. while shaking, was collected in asyringe and sufficiently homogenized. The platelet product was thenfiltered in the same manner as in Example 13, provided that theconcentrated platelet product was filled in the filter using a pump at aconstant linear velocity of 0.92 cm/min (flow rate: 1.2 ml/min) and,after suspending the flow of the concentrated platelet product for 30minutes, the platelet product was filtered at a constant linear velocityof 0.92 cm/min. The leukocyte removing capability and platelet recoveryrate were determined in the same manner as in Example 1. As a result,the leukocyte removing capability was 3.20 and the platelet recoveryrate was 88%.

The manner of operation and preferable conditions of blood filtrationusing the blood filtration apparatus of the present invention will bedescribed below.

EXAMPLE 20

When the first filtration method is carried out using the bloodfiltration apparatus shown in FIG. 1 in which a timer is used as asignal transmitting means (8), a flow rate controlling means (7,7′) isfirstly closed, and a blood storing unit (2) filled with blood is hungon a hook. After hooking the blood storing unit (2), a timer is set.After elapsing of the time previously set by the timer, the flow ratecontrolling means (7,7′) are released by an operation means (10) basedon signals from the timer to automatically start the blood to flow. Theblood from which leukocytes have been removed by flowing through aleukocyte-removing filter (4) is collected in a blood recovery unit (3).The period until the time when the flow rate controlling means (7,7′)are automatically released by the timer from the time when the bloodstoring unit (2) is hung on a hook is preferably not less than 5 minutesto less than 300 minutes, more preferably not less than 10 minutes toless than 180 minutes, and still more preferably not less than 30minutes to less than 120 minutes. If less than five minutes, asufficient blood cell concentration gradient may not be formed; if 300minutes or more, it is difficult to filter a large amount of blood.

When blood in which a blood cell concentration gradient has been formedby the centrifugal method is filtered, to inactivate the platelets thathave been activated by centrifugation, the timer is preferably set toautomatically release the flow rate controlling means (7,7′) at not lessthan 5 minutes to less than 180 minutes, and preferably not less than 10minutes to less than 90 minutes, after the centrifuged blood is hung ona hook.

EXAMPLE 21

When the second filtration method is carried out using the bloodfiltration apparatus shown in FIG. 1 in which a timer is used as thesignal transmitting means (8), the blood storing unit (2) filled withblood is hung on a hook. Next, the flow rate controlling means (7,7′)are opened to fill the leukocyte-removing filter (4) with blood. Whenthe leukocyte-removing filter (4) has been filled with blood, the flowrate controlling means (7,7′) are closed and the timer is set. Whenelapsing of the time previously set by the timer, the flow ratecontrolling means (7,7′) are released by the operation means (10) basedon signals from the timer to automatically start the blood to flow. Theblood from which leukocytes have been removed by flowing through theleukocyte-removing filter (4) is collected in the blood recovery unit(3).

The period of time for which the blood flow rate is controlled after thefilter has been filled with blood is preferably not less than 5 minutesto less than 300 minutes, more preferably not less than 10 minutes toless than 180 minutes, and still more preferably not less than 30minutes to less than 120 minutes. If the flow rate control time is lessthan five minutes, the effect of filling the filter with blood cannot beobtained and an increase in the platelet recovery rate may not beachieved. If 300 minutes or more, it is difficult to filtrate a largequantity of blood.

EXAMPLE 22

When the first filtration method is carried out using the bloodfiltration apparatus as shown in FIG. 2 in which an optical sensor isused as the signal transmitting means (9), a blood storing unit (2) isfilled with blood and hung on a hook, with the flow control means (7,7′)being maintained closed. When the optical sensor detects the bloodseparation degree set previously, the flow rate controlling means (7,7′)are automatically released by an operation means (10) based on signalsfrom the optical sensor to start the blood to flow. The blood from whichleukocytes have been removed by flowing through a leukocyte-removingfilter (4) is collected in the blood recovery unit (3). The blood cellseparation degree is preferably not less than 3% to less than 60%, andmore preferably not less than 10% to less than 45%. If the blood cellseparation degree is less than 5%, formation of the blood cellconcentration gradient is insufficient, whereby the filter performanceis not improved. If more than 60%, the hematocrit value in the lowerpart of the storing unit may excessively high, and an increase in thefiltration time, hemolysis due to an increased pressure loss, or aremarkable decrease in the flow rate may be potentially induced.

INDUSTRIAL APPLIABILITY

According to the blood filtration method of the present invention,leukocytes can be efficiently removed from a whole blood product. Inaddition, a high platelet recovery rate can be attained in a stablemanner while efficiently removing leukocytes from a whole blood productor platelet product. Since the blood filtration method of the presentinvention can highly remove leukocytes that cause transfusion sideeffects and can recover platelets as an effective component at a highyield, the method can be used as an effective method in bloodtransfusion medical treatment.

The blood filtration apparatus of the present invention is particularlysuitable for carrying out the blood filtration method of the presentinvention. Since the blood filtration apparatus of the present inventionstarts the filtration after the flow rate controlling means has beenautomatically released, it can relieve burden loaded on the operatorsand increase and stabilize the filter performance.

1. A blood filtration method for removing leukocytes from bloodcomprising: forming a blood cell concentration gradient in a storingunit for storing the blood; filtering all blood components in the orderof said blood cell concentration gradient in said storing unit throughonly one leukocyte-removing filter from a lower part of said storingunit; and collecting the filtered blood components in a recovery unit,wherein said blood cell concentration gradient is formed by allowing theblood to stand for a predetermined period of time, and wherein the bloodis a fresh whole blood product.
 2. The blood filtration method accordingto claim 1, wherein the blood cell concentration gradient is previouslyformed in the storing unit before filling the filter with blood.
 3. Theblood filtration method according to claim 1, wherein the blood cellconcentration gradient is formed by allowing the blood to stand for notless than 5 minutes to less than 300 minutes.
 4. The blood filtrationmethod according to claim 1, wherein the blood cell concentrationgradient is formed in the storing unit after filling the filter withblood.
 5. The blood filtration method according to claim 1, wherein theblood cell concentration gradient is formed by controlling the bloodflow rate for a predetermined period of time.
 6. The blood filtrationmethod according to claim 5, wherein the blood cell concentrationgradient is formed by controlling the blood flow rate over not less than5 minutes to less than 300 minutes.
 7. The blood filtration methodaccording to claim 1, wherein the blood is introduced into theleukocyte-removing filter from a lower part of the storing unit afterforming an erythrocyte concentration gradient so that the hematocritvalue of the blood in the lower part of the storing unit is larger thanthe hematocrit value of the whole blood product in the storing unit thathas been uniformly homogenized.
 8. The blood filtration method accordingto claim 7, wherein the hematocrit value of the blood in the lower partof the storing unit is not less than 1.05 times to less than 2.50 timesthe hematocrit value of the whole blood product in the storing unit thathas been uniformly homogenized.
 9. The blood filtration method accordingto claim 7, wherein the hematocrit value of the blood in the lower partof the storing unit is not less than 35% to less than 75%.
 10. The bloodfiltration method according to claim 1, wherein a blood cell separationdegree of the blood in the storing unit after forming the blood cellconcentration gradient is not less than 3% to less than 60%.
 11. Theblood filtration method according to claim 1, wherein leukocytes areselectively removed and platelets are allowed to pass through.